1. Field of the Invention
This invention relates to the purification of uricase and especially to the inactivation of catalase in a uricase preparation.
In human metabolism, there is a constant endogenous conversion of ingested nucleoproteins to substances such as purines. Purines, by a catabolic process, undergo further deamination and partial oxidation to uric acid, which, in humans, is normally excreted in the urine. This results in a nominal concentration of uric acid in the blood and urine of humans at all times. In pathological conditions, for example, renal insufficiency, uremia, gout and leukemia, there is an abnormal increase in the amount of uric acid found in urine or blood serum.
Medical science has recognized the usefulness of a test for measuring the amount of uric acid in the blood serum or urine as an aid in diagnosing the above-described conditions. One such test method involves the use of uricase to decompose uric acid by catalyzing the oxidation of uric acid to allantoin and hydrogen peroxide and measuring the amount of hydrogen peroxide formed, to determine the amount of uric acid
Uricase also can be used in pharmaceutical preparations. Such uricase must be substantially free of contaminants.
Animal organs have heretofore been the principal source of uricase. Difficulties in extraction and purification of uricase from such sources, along with the uncertainty of supply of such animal organs have encouraged the development of uricase production from microorganisms.
In the production of an enzyme such as uricase, whether by extraction from animal tissue or by fermentation of a microorganism, the desired enzyme is generally found in a liquid medium with various other macromolecules such as proteins, other enzymes and/or other undesirable material. Various methods of purifying uricase are well-known and include organic solvent precipitation, ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. Generally, even after such purification steps have been taken, the resulting uricase enzyme is not pure, but contains some amounts of protein impurities.
The principal contaminant of uricase is the enzyme catalase. Catalase is an undesirable impurity, whether the uricase is to be used as a pharmaceutical preparation or used for the quantitative analysis of uric acid in a detection system which includes hydrogen peroxide. Catalase causes the decomposition of hydrogen peroxide; thus the presence in uricase of catalase in an active form will produce a misleadingly low value of uric acid in the biological fluid measured.
2. Description of the Prior Art
U.S. Pat. No. 2,878,161 describes obtaining uricase from uricase-bearing tissue such as liver tissue derived from hogs, cattle or sheep.
The production of uricase from various microorganisms, including bacteria, fungi, and yeast is described in U.S. Pat. Nos. 3,431,176; 3,475,276; 3,620,923; 3,669,843; and 4,062,731.
U.S. Pat. No. 3,810,820 describes the production of uricase using bacteria and fungi belonging to various species. The mycelial extract obtained from the culture medium, containing uricase together with numerous proteins, is purified by a series of precipitations from aqueous medium using organic liquids miscible with water or aqueous solutions containing ammonium sulfate. The patentee suggests the use of adsorption upon hydroxyapatite, bentonite and alumina and subsequent extraction, followed by elution using saline solutions. Purification can be carried still further by subjecting the thus-obtained products to chromatography.
U.S. Pat. No. 4,064,010 describes a method for purification of uricase, and specifically the removal of catalase from uricase. The patentees refer to the use of ammonium sulfate as a purification technique and claim the process of separating catalase present by passing the catalase-containing uricase through a chromatographic column of cyanogen bromide-activated polysaccharide material having a hydrophobic ligand attached.
The complicated and expensive prior art methods indicate that there is a need for a simple and economical method for purification of uricase.